About one-third of breast cancer patients have estrogen receptor alpha-negative disease. Another group of breast cancer patients lose expression of their tumor's receptor during the course of disease progression. Compared to patients with estrogen receptor positive tumors, these patients do not respond to endocrine therapy, and tend to have more aggressive disease with earlier recurrence, resulting in less favorable prognosis. We have recently shown that by applying to cultured human breast cancer cells a methodology for chemical synthetic lethality screening, previously established in our laboratory in human and mouse cells, one can retrieve compounds that are synergistic lethal with ERalpha-deficiency. It is the subject of this application, to develop the methodology and the tools to a level that would enable the HTS of chemicals and the identification of synthetic lethal compounds in this medically important system. The specific aims of the project are: (i) To determine the dose response for three chemicals mediating ERa-episomal plasmid retention, (ii) To determine the false negative rate of these compounds, (iii) To test the false positive rate under conditions of medium alone, as compared to medium supplemented with unrelated bioactive molecules, (iv) To develop the statistical procedures for the HTS. (v) To screen by this method a diverse collection of approximately 750 bioactive molecules, (vi) To perform a counter screen to rule out artifacts, (vii) To evaluate the "hit" compounds by an independent assay, testing for differential toxicity as related to the genetic milieu. The poor prognosis of breast cancer patients with estrogen receptor a-negative disease calls for development of a selective, non-toxic adjuvant systemic therapy for this group. If successful, this novel approach will allow the screening of thousands of compounds derived from synthetic or natural resources, to identify chemicals selectively lethal to ERalpha-deficient tumor cells. This test case could then stimulate the dissemination of this methodology, resulting in wider spectrum of gene-related drugs towards other human diseases as well. Moreover, it should enable the implementation of the multigene shRNA expression libraries to this system, leading to the identification of gene targets synthetic lethal with ERa-deficiency in breast carcinomas.